Substance for restoring normal co-expression and interaction between the lox and nrage proteins

ABSTRACT

The invention relates to the use of a substance for restoring normal co-expression and interaction between the LOX and NRAGE proteins. The invention relates in particular to the use of an effective amount of at least one substance that modulates the expression and/or activity of the LOX of SEQ ID NO: 1 and/or that modulates the expression and/or activity of the NRAGE of SEQ ID NO: 2 for modulating the interaction between the LOX and NRAGE proteins to regulate the balance between the cellular proliferation, differentiation and apoptosis, particularly in cases where the balance between these phenomena is disturbed, and especially in cases where the interaction between LOX and NRAGE is absent or altered. The invention makes it possible to treat skin ageing, lichen planus, graft-versus-host reaction (GVH), eczema, psoriasis and a cancer, particularly an epithelial cancer and more particularly a cutaneous epithelial cancer of basocellular or spinocellular type.

STATE OF THE ART 1) Background

In multicellular organisms, homeostasis is maintained by a balancebetween cell proliferation, cell differentiation towards a definedfunction, and programmed or unprogrammed cell death.

The skin, for example, can be considered as an organ of which onespecific function is to protect the organism from external aggressions.Impermeability to the outside is assured by a highly keratinized corneallayer composed of dead cells. The rhythm of the life of epidermal cells,or keratinocytes, is therefore governed by a succession of changesleading to this differentiation into keratinized and dead cells: (1)maintenance of initiator cells (“stem” cells of the basal cell layer ofthe epidermis), (2) asymmetric division (each stem cell giving twosister cells), (3) differentiation (formation of the suprabasal celllayers of the epidermis from one of the “initiator sister” cells), (4)acquisition of a resistance to apoptotic cell death, (5) change intocornified cells, and (6) programmed cell death of the upper cell layersof the epidermis. Any modification of this balance can be pathological.

Those skilled in the art are familiar with several biological mechanismsand molecules which take account of certain aspects of the cellularphenomena of proliferation, differentiation and apoptosis. However, nodata give any real information about any one mechanism which would linkthese three phenomena and which would thus constitute a target on whichto act with a view to controlling and regulating them.

2) LOX (Protein)

LOX belongs to the family of the lysyl oxidases (LOs), which are amineoxidases dependent on copper. 5 LO genes have been characterized todate: LOX LOXL, LOXL2, LOXL3 and LOXL4. Where LOX is known for its rolein the crosslinking of collagen ex vivo and in vivo, LOXL is clearlyassociated with the homeostasis of elastic fibers, The in vivo role ofthe other isoforms is not known.

LOs are synthesized by a variety of cells such as fibroblasts, smoothmuscle cells, endothelial cells and keratinocytes. This enzyme istherefore present in numerous tissues such as the skin, the liver, thekidneys, the spleen and the aorta, at both extracellular andintracellular levels.

2.1) Stabilization of the Extracellular Matrix—Extracellular Role

The extracellular role of LOX, which is now well known, consists instabilizing the extracellular matrix (ECM) of connective tissues. Theactual function of LOX is to crosslink fibrillar collagens and elastin.To do this, it catalyzes the oxidative deamination of lysyl andhydroxylysyl residues of fibrillar procollagen molecules andtropoelastin, this reaction being accompanied by the release of ammoniaand hydrogen peroxide. The aldehyde residues formed then condensespontaneously with adjacent aldehyde or amine groups to formintramolecular and intermolecular bonds. These condensation reactionsgive rise to the bridging found in collagen and elastin fibers. LOX andLOs are therefore studied in biomedical fields that involve a change inthe ECM (ageing, fibroses, cancer, healing, osteoarticular andcardiovascular diseases, angiogenesis).

It has been demonstrated in vitro that organic nitriles are irreversibleLOX inhibitors. Thus β-aminopropionitrile (β-APN) binds to the activesite of LOX in competition with alkylamine substrates; LOs use β-APN assubstrate, thereby forming a Schiff base but without releasing thealdehyde product, which blocks the active site covalently without havingan effect on synthesis. On this basis the inventors have described theuse of different lysyl oxidase inhibitors, including β-APN, for avoidingthe dedifferentiation of certain cell types (chondrocytes, etc.) whichoccurs systematically when these cells are cultivated (Farjanel et al.,French patent 01.10443, CNRS, Use of lysyl oxidase inhibitors for cellculture and tissue engineering, filed on 3 Aug. 2001, publication no.: 2828 206). However, said document relates to lysyl oxidases as a wholeand not specifically to the LOX isoform; furthermore, it relatesexclusively to inhibition of the dedifferentiation of cells cultivatedin vitro, providing no teaching on the role of lysyl oxidases, or afortiori of LOX, in maintenance of the balance between proliferation,differentiation and apoptosis, nor does it provide any teaching on novelpartners or substrates for lysyl oxidases or LOX.

2.2) Intracellular Role

The intracellular role of LOX, and of LOs in general, is less wellknown. Thus, although it is acknowledged that LOX participates in theregulation of cell development, differentiation, mobility or senescence(Csiszar, Lysyl oxidases: A novel multifunctional amine oxidase family,Nucleic Acid Research and Molecular Biology, 2001, 70, 2-28), theunderlying molecular mechanisms are not elucidated.

2.2.1) LOX and Cellular Homeostasis

The possible involvement of LOX in the regulation of cellularhomeostasis (maintenance of a physiological balance betweenproliferation, differentiation and apoptosis) is commonly accepted (Jeayet al., Lysyl oxidase inhibits Ras-mediated transformation by preventingactivation of NF-KB, Mol. Cell. Biol., 2003, 23, 2251-2263), but theunderlying mechanisms are still unknown to those skilled in the art.

At the present time, those skilled in the art are thus in possession ofa few data indicating a possible relationship between LOX anddifferentiation, on the one hand, and LOX and cellproliferation/transformation, on the other. However, the prior art doesnot refer to a possible relationship between LOX and apoptosis.

2.2.2) LOX and Epidermal Differentiation

LOX has been located in the epidermis, its expression being regulated asa function of the level of differentiation of keratinocytes (Noblesse etal., Lysyl oxidase-like and lysyl oxidase are present in the dermis andepidermis of a skin equivalent and in human skin and are associated toelastic fibers, J. Invest. Dermatol., 2004, 122, 621-630). However, thestudies carried out hitherto have not made it possible to define therole of LOX in keratinocytes or to search for its partners in thesecells, which make little or no collagen or elastin.

2.2.3) LOX and Cell Transformation

The LOX gene is clearly associated with maintenance of the non-tumoralphenotype of cells.

Two hypotheses have been put forward to explain this role of LOX. Thefirst hypothesis suggests that crosslinking of the ECM induces athree-dimensional environment that favors maintenance of the non-tumoralstate. This hypothesis is supported by the fact that LOX and LOXL are nolonger expressed when cancers become invasive, whereas they are presentin cancers in situ (Peyrol et al., Lysyl oxidase gene expression in thestromal reaction to in situ and invasive ductal breast carcinoma, Am. J.Pathol. Feb., 150(2), 497-507, 1997). The other hypothesis concerns theintracellular roles of LOs on substrates which those skilled in the artare yet to discover (Li et al., Localization and activity of lysyloxidase within nuclei of fibrogenic cells, Proc. Natl. Acad. Sci. USA,1997, Nov. 25, 94(24), 12817-22).

It has in fact been shown that the LOX enzyme is a suppresser of the rasoncogene and that somatic mutations in the gene are associated withvarious cancers (Contente et al., Expression of gene rrg is associatedwith reversion of NIH 3T3 transformed by LTR-c-H-ras, Science, 1990,249, 796-798; Csiszar et al., Somatic mutations of the lysyl oxidasegene on chromosome 5q23.1 in colorectal tumors, Int. J. Cancer, 2002,97, 636-642). Recent studies show that the low level of expression ofLOX in fibroblasts transformed by ras is due to the activity of theFGF-2 autocrine growth factor and that the antitumoral drug suraminemakes it possible to reinduce the expression of the enzyme(Palamakumbura et al., Autocrine growth factor regulation of lysyloxidase expression in transformed fibroblasts, J. Biol. Chem., 2003,Aug. 15, 278(33), 30781-7; Epub 2003 Jun. 4). It has been shownelsewhere that the re-expression of LOX in these fibroblasts inhibitstheir growth in soft agar by acting on the NF-kB signaling pathway viaregulation of the location of the AKT protein (Jeay et al., Lysyloxidase inhibits Ras-mediated transformation by preventing activation ofNF-B, Mol. Cell. Biol., 23, 2251-2263, 2003).

As in the case of other tumor suppressors, LOX probably acts accordingto the availability and regulation of its cellular substrates andpartners, but the latter are still unknown to those skilled in the art.

2.2.4) Apoptosis

The term apoptosis is used to describe a particular form of cell deathwhose morphological characteristics differ from those of necrosis.

Apoptosis is a process of programmed cell death that requires theacquisition of caspases. In the course of this process the cells acquireparticularly remarkable morphological characteristics, such ascondensation of the chromatin and fragmentation of the nucleus, leadingto their self-destruction and their elimination from the tissue withoutdamaging the adjacent cells.

Apoptosis corresponds to the natural death of cells in the course oftheir development or during homeostasis.

The prior art provides information about a number of agents andmechanisms that are involved in regulating the cell cycle via thecellular processes of apoptosis and differentiation,

3) NRAGE (Protein)

Thus we are particularly familiar with one family of proteins that areinvolved in regulating the cell cycle, differentiation and apoptosis. Itis the family of the MAGE (melanoma associated antigen) proteins, one ofwhose members, the NRAGE protein, which is also called“neurotrophin-receptor-interacting MAGE homolog”, is particularly knownfor its proapoptotic role via the neurotrophic factor NGF.

The NRAGE protein comprises 778 amino acids. In the central region itcarries a first domain characteristic of the MAGE proteins: the MAGEhomology domain (MHD-1), and in the N-terminal region it carries asecond domain: MHD-2, present only in certain isoforms. These twodomains have zones rich in lysyl residues. Between these two domainsthere is a region called IRD (interspersed repeat domain), which doesnot exist in any other protein currently known,

The NRAGE protein is involved in controlling apoptosis via differentpathways.

By interacting with p75NTR, which is the “low affinity” receptor ofneutrophilic factors (NGF) or TNF, NRAGE can block the progression ofthe cell cycle and thus is proapoptotic via the caspase pathway.

NRAGE is also proapoptotic by interacting with cytoplasmic inhibitors ofapoptotic proteins, IAP.

NRAGE can also act directly on the activity of nuclear homeo factors,such as the factors Msx and Dix, which take part in the morphogenicregulation of tissues.

Although the expression of NRAGE is ubiquitous, the prior art does notrefer to its presence in the skin, nor does it provide any data givinginformation about a possible relationship between LOX and apoptosis orabout a link between LOX and NRAGE.

4) Conclusion on the Prior Art

The prior art does not provide the identity of novel partners orsubstrates for LOX, especially those taking part in maintenance of thecellular balance between proliferation, differentiation and apoptosis;moreover, the prior art does not provide the identity of these novelpartners or substrates either in epithelial cells (particularlykeratinocytes) or in any other cell type.

The prior art gives no information about the possible variations in theexpression of LOX in the epidermis (especially LOX expressed bykeratinocytes) as a function of age or of the existence or non-existenceof exposure to UV or other types of aggression, or in the case ofdiseases affecting the skin (psoriasis, graft-versus-host reaction,cancers, etc.).

The prior art does not describe the involvement of LOX in apoptosis.

The prior art does not provide information about the possible presenceof NRAGE in the skin, whether it be healthy skin, skin altered by age orUV or subjected to other types of aggression, or diseased skin.

The prior art does not offer known models for studying NRAGE in cells ofepithelial origin or in the epidermis.

The prior art provides no information about any kind of interactionbetween LOX and NRAGE, irrespective of the tissue.

The prior art does not provide a model for identifying active principlescapable of modulating the expression of LOX and/or NRAGE inkeratinocytes.

Furthermore, animal experimentation is currently banned in Europe forcertain applications and human experimentation is the subject of ethicaldebate. It is therefore unacceptable for the inventors to implement ascreening method using animals or humans.

In the three-dimensional model MIMESKIN® (Coletica, France), LOX isexpressed in the epidermis, its expression being regulated as a functionof the level of differentiation of the keratinocytes (Noblesse et al.,2004). These studies did not include research into the expression ofNRAGE or into the existence of a possible relationship between LOX andapoptosis, so it was not obvious that those skilled in the art would beinterested in modulating the expression of LOX and/or NRAGE in order toregulate cellular homeostasis, based on the balance betweenproliferation, differentiation and apoptosis, in cases where it isdisturbed (age, stress, disease), which thus constitutes a noveltechnical problem.

The prior art therefore fails to provide active principles capable ofmodulating the expression of intracellular partners for LOX (such asNRAGE), which may or may not be associated with modulation of theexpression of LOX for the purpose of acting on cell regulation. In thiscontext it is also very difficult to obtain objective criteria forjudging the impact of these active principles.

OBJECTS OF THE INVENTION

The main object of the invention is to solve the technical problemsreferred to above, especially the technical problem relating to theprovision of a method of identifying active principles for improving theinteraction between the LOX and NRAGE proteins in cases where theinteraction between LOX and NRAGE is absent or altered.

In particular the aim of the invention is to regulate the cellularbalance between proliferation, differentiation and apoptosis in caseswhere it is disturbed, as in the case of skin altered by age or UV orsubjected to other types of aggression, and/or in the case ofpathological situations such as psoriasis, eczema, graft-versus-hostreaction, lichen planus and/or cancerous diseases.

The invention further relates to the use of an active principle thatmodulates the expression of LOX and/or NRAGE for regulating the cellularbalance between proliferation, differentiation and apoptosis,particularly in cases where it is disturbed, as in the case of skinaltered by age or UV or subjected to other types of aggression, and/orin the case of pathological situations such as psoriasis, eczema,graft-versus-host reaction, and cancerous scars and/or diseases. Inparticular the invention relates to active principles for improving theinteraction between the LOX and NRAGE proteins in cases where theinteraction between LOX and NRAGE is absent or altered.

The invention makes it possible to solve the technical problem thatconsists in providing a method of locating the expression of NRAGE andfollowing this expression, especially on a cellular model of skin. Theinvention makes it possible to solve the technical problem that consistsin providing a method of diagnosing a state associated with poorregulation of the interaction between LOX and NRAGE.

DESCRIPTION OF THE INVENTION

The meanings of the terms used throughout the description, as understoodby the inventors, are given below:

“LOX”: the isoform of the human protein lysyl oxidase, LOX, as definedin particular by the amino acid sequence SEQ ID no. 1;

“NRAGE”: the human protein NRAGE as defined in particular by the aminoacid sequence SEQ ID no. 2;

“modulation of the expression of LOX”: modulation of the gene coding forLOX, especially modulation of the expression of the messenger RNA codingfor LOX, but also modulation of the synthesis of LOX from this messengerRNA, as well as modulation of the activity of LOX;

“modulation of the expression of NRAGE”: modulation of the gene codingfor NRAGE, especially modulation of the expression of the messenger RNAcoding for NRAGE, but also modulation of the synthesis of NRAGE fromthis messenger RNA, as well as modulation of the biological effect ofNRAGE.

These modulations must make it possible to reinduce a state of balancebetween proliferation, differentiation and apoptosis in situations wherethis balance is disturbed.

Active principles which are considered to be effective on LOX arepreferably those affording a difference of about ±50% in the expressionof the mRNA of LOX and/or a difference of about ±15% in the expressionof LOX and/or the activity of LOX on a model, comprising at least onecell type exhibiting LOX expression and/or activity, in contact withthese active principles, compared with the level of LOX expressionand/or activity in a control model (generally without being brought intocontact with the active principles).

Active principles which are considered to be effective on NRAGE arepreferably those affording a difference of about ±50% in the expressionof the mRNA of NRAGE and/or a difference of about ±15% in the expressionof NRAGE and/or the biological effect of NRAGE on a model, comprising atleast one cell type exhibiting NRAGE expression and/or activity, incontact with these active principles, compared with the level of NRAGEexpression and/or activity in a control model (generally without beingbrought into contact with the active principles).

Thus, according to a first feature, the present invention relates to theuse of an effective amount of at least one substance that modulates theexpression and/or activity of at least the LOX protein of sequence IDno. 1, and/or that modulates the expression and/or activity of at leastthe NRAGE protein of sequence ID no. 2, for the manufacture of acomposition for modulating the interaction between LOX and NRAGE inorder to regulate the cellular balance between proliferation,differentiation and apoptosis, particularly in cases where the balancebetween these phenomena is disturbed, and especially in cases where theinteraction between LOX and NRAGE is absent or altered.

The invention relates to the use of an effective amount of at least onesubstance that modulates the expression and/or activity of LOX ofsequence ID no. 1, and/or that modulates the expression and/or activityof NRAGE of sequence ID no. 2, for the manufacture of a composition forimproving the interaction between the LOX and NRAGE proteins in caseswhere the interaction between LOX and NRAGE is absent or altered.

The invention further relates to the use of an effective amount of atleast one substance that modulates the expression and/or activity of atleast the NRAGE protein of sequence ID no. 2, and that optionallymodulates the expression and/or activity of the LOX protein of sequenceID no. 1, for the manufacture of a composition for preventing ortreating at least one state in which the cellular balance betweenproliferation, differentiation and apoptosis is absent or altered.

Advantageously, the expression of LOX and/or NRAGE is modulated inepithelial cells, particularly in keratinocytes.

Advantageously, the purpose of said substance is to modulate theinteraction between the LOX protein and the NRAGE protein, saidinteraction occurring especially in the IRD domain of the NRAGE protein.

Advantageously, the interaction between LOX and NRAGE comprises thepolymerization of NRAGE, particularly its dimerization, by LOX-inducedenzymatic catalysis.

Advantageously, the interaction between LOX and NRAGE involves theproduction of H₂O₂ resulting from the catalytic activity of LOX, theH₂O₂ then activating other molecules, for example neutralsphingomyelinase or NF-kB.

Advantageously, the interaction between LOX and NRAGE involves anon-enzymatic activity of LOX, particularly an activity exerted by thepro-region (A22-D169) of LOX.

Advantageously, the purpose of the substance is to treat and/or preventa condition selected from the group consisting of exposure of cells to astress, particularly exposure of cells to heat, or exposure of cells toradiation, particularly solar radiation, or exposure of cells to a toxicagent, for example a chemical or microbiological agent, skin ageing,lichen planus, graft-versus-host reaction (GVH), eczema, psoriasis and acancer, particularly an epithelial cancer.

Advantageously, the purpose of the substance is to reduce cellproliferation in the case of hyperproliferation, particularly in acancer, more particularly an epithelial cancer and most particularly acutaneous epithelial cancer, of basocellular or spinocellular type,psoriasis or eczema.

Advantageously, the purpose of the substance is to reduce apoptosis inthe epidermis in the case of substantial apoptosis, particularly duringskin ageing, exposure of the skin to a stress, particularly exposure toheat, or exposure of the skin to radiation, particularly solarradiation, or exposure of the skin to a toxic agent, for example achemical or microbiological agent, or graft-versus-host reaction (GVH).

Advantageously, the purpose of the substance is to increase cellproliferation in the case of cell hypoproliferation in the epidermis,particularly during ageing, exposure of the skin to heat, exposure ofthe skin to radiation, particularly solar radiation, orgraft-versus-host reaction (GVH).

Advantageously, the purpose of the substance is to stimulate theexpression of LOX, and optionally to inhibit the expression of NRAGE,during skin ageing, exposure of the skin to a stress, particularlyexposure to heat, or exposure of the skin to radiation, particularlysolar radiation, or exposure of the skin to a toxic agent, for example achemical or microbiological agent, or graft-versus-host reaction (GVH).

Advantageously, the purpose of the substance is to stimulate theexpression and/or activity of NRAGE, and optionally to inhibit theexpression and/or activity of LOX, in the epidermis in order to preventor treat psoriasis or eczema.

Advantageously, the purpose of the substance is to stimulate theexpression of LOX and NRAGE in order to prevent or treat a cancer,particularly an epithelial cancer and more particularly a cutaneousepithelial cancer, of basocellular or spinocellular type, or lichenplanus.

Advantageously, said composition is a cosmetic, neutraceutical,dermo-pharmaceutical or pharmaceutical composition.

Advantageously, the cellular balance between proliferation,differentiation and apoptosis is the balance between proliferation,differentiation and apoptosis of the keratinocytes.

Advantageously, the starting material used to prepare the activeprinciples, in the case of plants (preferably roots, stems, barks,flowers, fruits, seeds, germs, gums, exudates, leaves or whole plants)or proteins, may or may not be sterilized by radiation, for example betaor gamma radiation preferably at a dose of 5 kGy, and is then reduced topowder if necessary, for example by grinding at room temperature. Thepowder is then e.g. dispersed at a rate of 2 to 5% (weight/weight) ofpowder, preferably 5%, either in a polar solvent, for example water, analcohol, a glycol such as butylene glycol, or a polyol, and/or a mixtureof polar solvents, advantageously a mixture of water with an alcohol,glycol or polyol (such as ethanol, glycerol, butylene glycol and otherglycols, xylitol, etc.) in variable proportions, preferably a 75/25 or50/50 water/butylene glycol mixture, or in an apolar solvent, forexample an alkane, or a mixture of apolar solvents, or in a mixture ofpolar and apolar solvents. Preferably after stirring, e.g. magneticstirring, for at least 2 hours, and optionally heating of the solvent,the sample is preferably clarified by decantation or centrifugation andthen filtered, preferably on a 0.45 μm or 0.22 μm filter.

Advantageously, the starting material used to prepare the activeprinciples, in the case of characterized molecules (e.g. moleculesobtained by synthesis or hemisynthesis, biological molecules obtained bypurification), is diluted in a solvent, preferably water or dimethylsulfoxide (in a concentration preferably of between 10⁻⁶ M and 10⁻² Mand particularly preferably in the order of 10⁻⁴ M, or preferably ofbetween 1% weight/weight and 5% weight/weight, depending on themolecules). The solution obtained is then optionally filtered,preferably on a 0.45 μm or 0.22 μm filter.

Advantageously, the active principles obtained by one of the methodsdescribed above are used in a final concentration preferably of between0.01% volume/volume (v/v) and 10% (v/v) and particularly preferably ofbetween 0.1% and 1% (v/v).

Advantageously, said substance is selected from the group consisting ofa soya extract, an ephedra extract, a hop extract and a cinnamonextract.

According to a second feature, the invention relates to a method ofidentifying at least one active principle described above, the purposeof which is in particular to modulate the interaction between LOX andNRAGE in order to prevent or treat at least one state in which thecellular balance between proliferation, differentiation and apoptosis isabsent or altered, characterized in that it comprises:

bringing the active principle into contact with at least one type ofliving cell capable of expressing the LOX protein (SEQ ID no. 1) and/orthe NRAGE protein (SEQ ID no. 2), and

analyzing the expression of LOX and/or NRAGE, especially to identify anactive principle that modulates the expression and/or activity of LOXand/or NRAGE in order to improve the cellular balance betweenproliferation, differentiation and apoptosis.

The invention further relates to a method of identifying at least oneactive principle for modulating the interaction between LOX and NRAGE inorder to improve the interaction between the LOX and NRAGE proteins incases where the interaction between LOX and NRAGE is absent or altered,characterized in that it comprises:

bringing the active principle into contact with at least one type ofliving cell capable of expressing the LOX protein (SEQ ID no. 1) and/orthe NRAGE protein (SEQ ID no. 2), and

analyzing the expression of LOX and/or NRAGE, especially to identify anactive principle that modulates the expression and/or activity of LOXand/or NRAGE in order to improve the interaction between the LOX andNRAGE proteins in cases where the interaction between LOX and NRAGE isabsent or altered.

Advantageously, the balance between the proliferation, differentiationand apoptosis of said living cells capable of expressing the LOX and/orNRAGE protein, or the interaction between the LOX and NRAGE proteins, isabsent or altered before they are brought into contact with the activeprinciple.

Advantageously, the living cells are epithelial cells, particularlykeratinocytes.

Advantageously, the method comprises analyzing the expression of themessenger RNAs of LOX and/or NRAGE.

Advantageously, the method comprises using quantitative RT-PCR with thefollowing primers in particular:

for the LOX gene:

Sense ACGTACGTGCAGAAGATGTCC Antisense GGCTGGGTAAGAAATCTGATG

for the NRAGE gene:

Sense TGCACAGACATCAGCAGATGG Antisense TTCACGGATGATATCTCTCAGC

Advantageously, the method comprises analyzing the kinetics ofexpression of the messenger RNAs, for example by quantitative RT-PCR.

The invention further relates to a method for the cosmetic care ortherapeutic treatment of a subject in whom the balance between thecellular phenomena of proliferation, differentiation and apoptosis in atleast one cell type is disturbed, said method comprising improving theinteraction between LOX and NRAGE by the application or administrationof a substance that modulates the expression of LOX and/or NRAGE.

The invention further relates to a method for the cosmetic care ortherapeutic treatment of a subject in whom the interaction between LOXand NRAGE is absent or altered, said method comprising the applicationor administration of a substance that modulates the expression and/oractivity of LOX of sequence ID no. 1, and/or that modulates theexpression and/or activity of NRAGE of sequence ID no. 2.

The invention further relates to a method for the cosmetic care ortherapeutic treatment of a subject presenting with a state in which thecellular balance between proliferation, differentiation and apoptosis isabsent or altered, said method comprising the application oradministration of a substance that modulates the expression and/oractivity of NRAGE, and optionally that modulates the expression and/oractivity of LOX.

According to a third feature, the invention relates to a process for thepreparation of a composition, comprising:

the use of a previously defined method of identification for identifyingan active principle that modulates the expression and/or activity of LOXand/or NRAGE in order to improve the cellular balance betweenproliferation, differentiation and apoptosis, and

the mixing of the active principle with at least one excipient in orderto produce a cosmetic, neutraceutical, dermopharmaceutical orpharmaceutical composition for preventing or treating at least one statein which the cellular balance between proliferation, differentiation andapoptosis is absent or altered.

The invention further relates to a process for the preparation of acomposition, comprising:

the use of a previously defined method of identification for identifyingan active principle that modulates the expression and/or activity of LOXand/or NRAGE in order to improve the interaction between the LOX andNRAGE proteins, and

the mixing of the active principle with at least one excipient in orderto produce a cosmetic, neutraceutical, dermopharmaceutical orpharmaceutical composition for improving the interaction between the LOXand NRAGE proteins in cases where the interaction between LOX and NRAGEis absent or altered.

According to a fourth feature, the invention relates to a method oflocating NRAGE, comprising the use of at least one anti-NRAGE antibodyfor detecting and locating the presence of NRAGE, particularly in amodel of reconstructed skin comprising at least keratinocytes, or asection of skin, preferably a section of skin originating from a personwhose epidermis exhibits a state in which the cellular balance betweenproliferation, differentiation and apoptosis is absent or altered.

According to a fifth feature, the invention relates to the use of ananti-NRAGE antibody for the preparation of a composition for detecting amodulation of the expression of NRAGE at the cellular level,particularly in epithelial cells and preferably in keratinocytes.

Advantageously, the purpose of the composition is to detect a state inwhich the cellular balance between proliferation, differentiation andapoptosis, or the interaction between LOX and NRAGE, particularly in theepidermis, is absent or altered, said state being selected from thegroup consisting of exposure of cells to a stress, particularly exposureof cells to heat, or exposure of cells to radiation, particularly solarradiation, or exposure of cells to a toxic agent, for example a chemicalor microbiological agent, skin ageing, lichen planus, graft-versus-hostreaction (GVH), eczema, psoriasis and a cancer, particularly anepithelial cancer and more particularly a cutaneous epithelial cancer,of basocellular or spinocellular type.

Advantageously, the composition also comprises an anti-LOX antibody fordetecting a modulation of the expression of LOX at the cellular level,particularly in epithelial cells and preferably in keratinocytes.

Advantageously, the composition also comprises an anti-LOX antibody fordetecting a modulation of the expression of LOX in keratinocytes.

Advantageously, the keratinocytes are human keratinocytes.

DETAILED DESCRIPTION OF THE INVENTION:

Unexpectedly, the inventors have discovered an interaction between LOXand the NRAGE protein. This discovery is fundamental and is the startingpoint of the present invention. It actually shows LOX to be the proteinwhich, being present at the intersection of the proliferation,differentiation and apoptosis pathways, is capable of controllingcellular homeostasis.

The inventors have also discovered that the NRAGE protein is present inparticular in the epidermis and is expressed in particular by thekeratinocytes. This discovery also enables the interaction between LOXand NRAGE to be made an action target for cosmetic care or treatmentsfor restoring cellular homeostasis in situations where it is disturbed.

1) Discovery and Characterization of the LOX/NRAGE Interaction In Vitro

Having previously discovered the presence of LOX in the epidermis(Noblesse et al., Lysyl oxidase-like and lysyl oxidase are present inthe dermis and epidermis of a skin equivalent and in human skin and areassociated to elastic fibers, J. Invest. Dermatol., 122, 621-630, 2004),the inventors endeavored to determine the role(s) that LOX might play atthis level, in the knowledge that its main currently acknowledgedrole—crosslinking of collagen and elastin—was rather unlikely in thesecells, which make little or no collagen or elastin at the level whereLOX was located.

With this objective in mind, they searched for protein partners for LOXby the yeast two-hybrid technique and a library of normal human skinkeratinocyte cDNA was screened using the lure GAL4 BD-hLOXmat inparticular, thereby making it possible to identify NRAGE as a potentialpartner for LOX (cf. Example 1).

Using the two-hybrid technique in Hela cells, the inventors thenverified that this potential partnership was also observed in mammaliancells (cf. Example 2).

The next step consisted in determining whether the identification ofthis potential partnership between LOX and NRAGE could correspond to adirect physical interaction between these two proteins. This was carriedout in particular by a technique involving co-immunoprecipitation of theLOX and NRAGE proteins produced after transfection of their genes intoCos7 cells. The results obtained on the one hand confirmed the physicalinteraction between LOX and NRAGE and on the other hand made it possibleto specify that LOX interacted with a particular region of NRAGE calledIRD (interspersed repeat domain), which is a region specific to NRAGE inthe family of the MAGE proteins (cf. Example 3).

In this context it should be pointed out at this stage that thediscovery of the interaction between LOX and NRAGE is of very particularinterest when one considers, in combination, on the one hand that theNRAGE protein contains two domains rich in lysyl residues, which couldbe favorable sites for its dimerization under the catalytic action ofLOX, and on the other hand that it is precisely in the form of a dimerthat the functionality of NRAGE seems to manifest itself.

With this discovery of the existence of a direct physical interactionbetween LOX and NRAGE in vitro, located in the IRD region of NRAGE, theinventors asked themselves the question whether this interaction alsotook place in vivo. It is essential to recall at this stage that, incontrast to LOX, the presence of NRAGE in the skin had never previouslybeen described.

2) Identification of the Presence of NRAGE in Normal Human Skin andReconstructed Normal Human Skin

The inventors identified the expression of NRAGE in both the dermis andthe epidermis of the skin (cf. Examples 4 and 5).

Within the framework of the present invention the inventors used amethod of locating the expression of NRAGE in the skin.

In the epidermis the inventors demonstrated, unexpectedly, that NRAGEwas not expressed homogeneously—as would have been expected of a proteinwhose presence is generally described as ubiquitous—but in the form ofan expression gradient.

Reminder of the structure of the epidermis:

The epidermis is a stratified epithelium resting on a basal membranewhich anchors it to the dermis. Its thickness varies from 60 to 100 μmon average and it consists of 4 continuous layers, from the deepest tothe surface, resulting from a progressive differentiation of thekeratinocytes:

the basal cell layer (single row of cells)

the prickle cell layer (5 to 6 monocellular layers)

the granular layer (1 to 3 monocellular layers)

the horny layer (5 to 10 monocellular layers)

Thus NRAGE is absent in the basal cell layer and appears in the firstlayers of differentiated keratinocytes, the labeling increasing in thehorny layers. The labeling of the first suprabasal cell layers of theepidermis corresponds to that observed for LOX, which appears from thebasal cell layer upwards. On the other hand, the labeling of LOXdecreases in the upper layers of the epidermis, where NRAGE is present,so a distinction is made between the following 3 zones:

1 lower zone of the epidermis, comprising the basal cell layer and thefirst proliferative suprabasal cell layers, where only LOX is expressed;

1 intermediate zone extending from the first non-proliferativesuprabasal cell layers to the first monocellular horny layers, in whichLOX and NRAGE are co-expressed;

and 1 upper zone of the epidermis, where only NRAGE is expressed.

Observations at the cellular level show that LOX and NRAGE appear at theperiphery of the cell, particularly in the submembranous region, NRAGEalso appearing in the cytoplasm (cf. Examples 4 and 5).

The inventors thus identified, for the first time, the presence of NRAGEin the skin, both in the dermis and the epidermis. They also showed thatthe epidermis comprises a part in which both the LOX and NRAGE proteinsare expressed and share the same location at the cellular level,especially in the keratinocytes, said location being the submembranousperipheral zone (NRAGE also being present in the cytoplasm).

The inventors thus demonstrated fulfilment of the conditions for thedirect interaction they had identified in vitro to be able to take placein the epidermis in the zone where both LOX and NRAGE are located.

3) Identification of Disturbances of the Ex Ression of LOX and/or NRAGEin the Epidermis With Age and in Certain Pathological Situations

The inventors also demonstrated, unexpectedly, that skin ageing and anumber of pathological situations are accompanied by disturbances of theexpression of LOX and/or NRAGE in the epidermis.

3.1) State of LOX and NRAGE in the Epidermis

3.1.1) Skin of Elderly Persons

The skin of elderly persons is characterized by a hypoproliferativeepidermis that is very thin (reduced to a few cell layers) andhyperkeratinized.

The inventors identified the total absence of LOX (detectable by thetechniques used) in aged skin. On the other hand, NRAGE is stronglyexpressed, is located in the cytoplasm and appears from the firstsuprabasal cell layer upwards, without an expression gradient.

Thus the present invention makes it possible to stimulate the expression(and/or activity) of LOX, with or without inhibiting the expression(and/or activity) of NRAGE, so as to reinduce a regulateddifferentiation zone and/or apoptosis zone, in particular by restoring aLOX and NRAGE co-expression zone. This also thickens the skin,especially the epidermis.

Thus the purpose of the present invention is especially to correct orprevent the effects of skin ageing.

3.1.2) Graft-Versus-Host Reaction (GVH)

GVH is a disease which can arise following allografts of hemopoieticstem cells. It is associated with the effect of the immune cells(lymphocytes) contained in the graft on the patient's normal organs(especially the skin, liver and digestive tract). In the skin itmanifests itself in the form of a maculopapular, pruriginous andinflammatory eruption.

Patients suffering from this disease have very thin skin with a highlyapoptotic epidermis.

The histological study carried out by the inventors identified theabsence of LOX and a very pronounced presence of NRAGE, which is locatedin the cytoplasm and appears from the first suprabasal cell layersupwards.

Thus the present invention makes it possible to stimulate the expression(and/or activity) of LOX, with or without inhibiting the expression(and/or activity) of NRAGE, in order to reinduce a LOX and NRAGEco-expression zone, enabling them to interact and resulting in areduction in the cutaneous manifestations of GVH, this being accompaniedespecially by a thickening of the skin, particularly the epidermis.

Thus the present invention enables patients suffering from GVH to reducethe cutaneous manifestations of this disease.

3.1.3) Lichen Planus

Lichen is a skin disease of unknown cause which is characterized by thepresence of violet, flat, solid, dry and very pruriginous papules a fewmillimeters in diameter.

The inventors identified a very large decrease in the expression of LOX,or even its total absence, and a very irregular expression of NRAGE.These observations certainly reflect a non-existent or very weak levelof interaction between the two proteins studied.

Thus the present invention makes it possible to stimulate the expression(and/or activity) of LOX, with or without modulating the expression(and/or activity) of NRAGE, in order to reinduce a LOX and NRAGEco-expression zone, enabling them to interact and making it possible toreturn to a normal epidermal state.

Thus the present invention enables patients suffering from lichen planusto treat, reduce or prevent this disease.

3.1.4) Psoriasis

Psoriasis is a chronic skin disease characterized by erythematosquamouslesions. The fundamental feature of the disease is an increase in therate of multiplication of the keratinocytes, which is responsible for amore rapid renewal and a thickening of the epidermis.

Histologically one observes a hyperproliferation of the cells involvedin the first steps of terminal differentiation, i.e. an incompleteterminal differentiation, which is associated with the absence or veryweak presence of apoptosis.

The inventors detected a very strong expression of LOX and a moderatepresence of NRAGE with a more or less homogeneous labeling of the zonesin question, showing no expression gradient. These characteristics of anintensity of expression that varies from the norm are coupled with muchmore important anomalies affecting the location of the proteins inquestion. Thus, in psoriatic skin, LOX is essentially expressed in thelower part of the epidermis and NRAGE solely in its upper part, theexpression of the proteins thus being shifted without there being theoverlap zone normally observed in healthy skin. At the cellular levelNRAGE is observed only in the cytoplasm and not in the submembranousperipheral zone.

These observations show that, in psoriasis, although the LOX and NRAGEproteins are both present in the epidermis, they cannot interactdirectly with one another because they are not physically present in thesame place. This absence of interaction results in dysfunctions observedin the epidermis, thus reflecting the role of the interaction betweenLOX and NRAGE in maintaining epidermal homeostasis.

Thus inhibition of the expression (and/or activity) of LOX, and/oroptionally stimulation, preferably partial stimulation, of theexpression (and/or activity) of NRAGE, makes it possible in particularto obtain an overlap zone of LOX and NRAGE expression in order toreinduce a regulated proliferation zone that makes it possible to reducehyperproliferation by promoting apoptosis in the keratinocytes.

Furthermore, stimulation of the expression (and/or activity) of NRAGE,and/or inhibition, preferably partial inhibition, of the expression(and/or activity) of LOX, makes it possible in particular to obtain anoverlap zone of LOX and NRAGE expression in order to reinduce aregulated proliferation zone that makes it possible to reducehyperproliferation by promoting apoptosis in the keratinocytes.

Thus the present invention makes it possible to prevent and/or treatpsoriasis or reduce some of its effects.

3.1.5) Eczema

Eczema is a skin complaint characterized clinically by blotches, more orless extensive, localized swellings, and weeping vesicles thatsubsequently form scabs, accompanied by intense itching. In its chronicphase eczema is complicated by a modification of the skin withthickening. Apoptosis is reduced in the epidermis.

The inventors identified a very strong expression of LOX located at theperiphery of the cells. On the other hand, NRAGE is weakly expressed andis observed only in the cytoplasm, which certainly reflects anon-existent or very weak level of interaction between the two proteinsstudied.

Thus stimulation of the expression (and/or activity) of NRAGE, and/orinhibition, preferably partial inhibition, of the expression (and/oractivity) of LOX, makes it possible to increase apoptosis and thereby toreduce some of the effects of eczema.

Thus the present invention makes it possible to prevent and/or treateczema and in particular to reduce some of its effects.

3.1.6) Epithelial Skin Cancers

A distinction is made between two major types of epithelial skincarcinoma:

basocellular carcinoma (90% of cases) is an essentially local, slowlydeveloping tumor which virtually never metastasizes. It results from anuncontrolled proliferation of the keratinocytes of the basal cell layer;

spinocellular carcinoma (10% of cases) has a much more aggressive localdevelopment and can metastasize. It originates from an uncontrolledproliferation of the keratinocytes of the prickle cell layer.

The inventors identified the absence of LOX and NRAGE in the invasivecells of the two types of cancer studied (basocellular and spinocellularcancers), with a progressive loss of LOX and NRAGE in the epidermis inthe vicinity of the tumors. The inventors also observed that LOX isstrongly expressed in the stromal reaction around the tumors, whereasNRAGE is absent in this reaction.

It may be noted at this stage that the loss of expression of LOX, whichhad never previously been identified in the case of these two types ofcancer, is unexpected and is perhaps specific to epithelial cancers,since LOX is generally considered to be present in cancers in situ.

The loss of expression of NRAGE is completely novel as well and hasnever been described in any cancer.

Thus stimulation of the expression (and/or activity) of NRAGE and theexpression (and/or activity) of LOX makes it possible to restorehomeostasis. It is possible to bring about a reversion of the tumoralphenotype, especially in epithelial skin cancers.

3.2) Conclusion on the Studies on Pathological Tissues

On the basis of these observations the inventors were able todemonstrate that the situations involving a deregulation of the balancebetween proliferation, differentiation and apoptosis, whether this be inthe epidermis of elderly subjects or subjects suffering from differentdiseases affecting the epidermis in particular, were systematicallycharacterized by defects in the expression of LOX and/or NRAGE, saiddefects being likely to change their interaction or render itimpossible.

This finding led the inventors to search for a means of restoring thebalance between proliferation, differentiation and apoptosis byutilizing the control exerted by LOX or NRAGE, preferably by utilizingthe LOX-NRAGE couple.

Thus, on the basis of these unexpected discoveries, the inventorsprovided methods of identifying active principles that modulate theexpression of LOX and/or NRAGE in order to restore the control exertedby these proteins via their interaction, for the purpose of identifyingactive principles for the preparation of compositions, especiallycosmetic or pharmaceutical compositions.

In this way, in the case of epidermal hypoproliferation (aged skin,GVH), which is characterized by an extremely thin epidermis and theabsence of expression of LOX, the expression of LOX is stimulated, withor without inhibiting the expression of NRAGE, in order to reinduce, viamodulation of the co-expression of LOX and NRAGE, a regulateddifferentiation and apoptosis zone that leads to a thickening of theskin.

In the case of epidermal hyperproliferation (psoriasis, eczema), whereNRAGE is underexpressed, the expression of NRAGE is stimulated, with orwithout (slightly) inhibiting that of LOX, in order to promote apoptosisby reinducing a regulated proliferation zone (LOX/NRAGE overlap) andmaking it possible to stop the hyperproliferation.

In the case of a high level of apoptosis in the epidermis (aged skin,exposure of the skin to a stress, particularly exposure to heat, orexposure of the skin to radiation, particularly solar radiation, orexposure of the skin to a toxic agent, for example a chemical ormicrobiological agent, or graft-versus-host reaction—GVH), where NRAGEis overexpressed, the expression of NRAGE is inhibited, with or withoutstimulating the expression of LOX.

4) Identification of the Involvement of LOX in Cellular Apoptosis

Unexpectedly, the inventors identified the existence of a hithertounknown link between LOX and apoptosis. Thus the data obtained reflectthe antiapoptotic role of LOX in the keratinocytes, said role involvingregulation of the proapoptotic protein NRAGE in particular.

5) Search for Active Principles

Active principles were identified in particular by analyzing theexpression of the messenger RNAs of LOX and NRAGE, especially onkeratinocytes in culture and preferably on human keratinocytes. Theactive principles whose activity is to be tested are placed in contactwith the keratinocytes in culture for a sufficient time and underappropriate conditions for the contact to be effective. The activeprinciples are preferably tested in different concentrations so that anyinfluence of concentration can be detected.

Advantageously, the active principles screened in the present inventionare of vegetable origin, in particular so as to avoid the problemsassociated with chemical synthesis. The advantages of active principlesof vegetable origin are well known to those skilled in the art,especially in pharmacy, dermopharmacy, neutraceutics and cosmetics.

The search for active principles is carried out especially by extractingthe total RNAs and then performing quantitative RT-PCR. In particular,the preferred primer sequences are those used in Example 13, withoutimplying a limitation.

The amount of cDNA in each assay is plotted against the amount of actincDNA. The effect of the presence or absence of active principles is thencompared. If the expression and/or activity of NRAGE and/or LOX aremodulated (stimulated or inhibited) relative to the controls, thesubstance can then be qualified as an active principle. Advantageously,an active principle makes it possible to modulate the expression of themessenger RNA of LOX and/or NRAGE by at least 50% or the expressionand/or activity of LOX and/or NRAGE by at least 15%.

The compounds according to the present invention are prepared in theform of compositions, especially cosmetic, dermopharmaceutical orpharmaceutical compositions. The excipient for these compositionstherefore contains e.g. at least one compound selected from the groupconsisting of preservatives, emollients, emulsifiers, surfactants,moisturizers, thickeners, conditioners, matting agents, stabilizers,antioxidants, texturizing agents, brightening agents, film-formingagents, solubilizers, pigments, colorants, perfumes and sun filters.These excipients are preferably selected from the group consisting ofamino acids and their derivatives, polyglycerols, esters, cellulosepolymers and derivatives, lanolin derivatives, phospholipids,lactoferrins, lactoperoxidases, sucrose-based stabilizers, vitamin E andits derivatives, natural and synthetic waxes, vegetable oils,triglycerides, unsaponifiables, phytosterols, plant esters, siliconesand their derivatives, protein hydrolyzates, jojoba oil and itsderivatives, liposoluble/water-soluble esters, betaines, amine oxides,plant extracts, sucrose esters, titanium dioxides, glycines andparabens, and particularly preferably from the group consisting ofbutylene glycol, steareth-2, steareth-21, glycol-15 stearyl ether,cetearyl alcohol, phenoxyethanol, methylparaben, ethylparaben,propylparaben, butylparaben, butylene glycol, natural tocopherols,glycerol, sodium dihydroxycetyl phosphate, isopropyl hydroxycetyl ether,glycol stearate, triisononanoin, octyl cocoate, polyacrylamide,isoparaffin, laureth-7, a carbomer, propylene glycol, glycerol,bisabolol, a dimethicone, sodium hydroxide, PEG-30dipolyhydroxystearate, capric/caprylic triglycerides, cetearyloctanoate, dibutyl adipate, grapeseed oil, jojoba oil, magnesiumsulfate, EDTA, a cyclomethicone, xanthan gum, citric acid, sodiumlaurylsulfate, mineral waxes and oils, isostearyl isostearate, propyleneglycol dipelargonate, propylene glycol isostearate, PEG-8 beeswax,hydrogenated palm kernel glycerides, hydrogenated palm glycerides,lanolin oil, sesame oil, cetyl lactate, lanolin alcohol, castor oil,titanium dioxide, lactose, sucrose, low density polyethylene and anisotonic saline solution.

Advantageously, the abovementioned compositions are formulated in a formselected from the group consisting of an aqueous or oily solution, acream or an aqueous or oily gel, particularly in a pot or tube,especially a shower gel or a shampoo; a milk; an emulsion, microemulsionor nanoemulsion, particularly an oil-in-water, water-in-oil, multiple orsilicone emulsion; a lotion, particularly in a glass or plastic bottle,a dosing bottle or an aerosol; an ampoule; a syrup; a liquid soap; ahypoallergenic cleansing bar; an ointment; a foam; an injectablesolution; an anhydrous, preferably liquid, pasty or solid product, forexample in stick form, especially in the form of lipstick; a powder; anda tablet.

In the Figures:

FIG. 1 shows a sequence and a diagram of the NRAGE protein;

FIG. 2 shows the mean of the results obtained in Example 2 for thetwo-hybrid interaction;

FIG. 3 shows the identification of the presence of LOX and NRAGE inreconstructed skin;

FIG. 4 shows the location of LOX and NRAGE on human skin sections byconfocal microscopy;

FIG. 5 shows the detection of LOX and NRAGE on' human skin sections froma 91-year-old donor;

FIG. 6 shows the detection of LOX and NRAGE in the skin of a personsuffering from graft-versus-host reaction;

FIG. 7 shows the detection of LOX and NRAGE in the skin of a patientpresenting with a cancer of basocellular or spinocellular type;

FIG. 8 shows the detection of LOX and NRAGE in the skin of a patientsuffering from lichen planus;

FIG. 9 shows the identification of the location of LOX and NRAGE in theskin of a patient suffering from psoriasis;

FIG. 10 shows the identification of the location of LOX and NRAGE in theskin of a patient suffering from eczema.

FIG. 11 shows the global labelling of the reconstructed skin with Evansblue (red epidermal layers, blue cells, red fibers of the dermalsubstrate, dermo-epidermal junction in dotted lines).

FIG. 12 shows the immuno-histochemical detection of cyto-keratin 10 onthe reconstructed skin (cells strongly expressing the red label, thenuclei in blue, the dermo-epidermal junction in dotted lines).

FIG. 13 shows the immuno-histochemical detection of transglutaminase onthe reconstructed skin (cells strongly expressing the red label, thenuclei in blue, the dermo-epidermal junction in dotted lines).

Other objects, characteristics and advantages of the invention willbecome clearly apparent to those skilled in the art from the explanatorydescription referring to Examples, which are given solely by way ofillustration and cannot in any way limit the scope of the invention.

The Examples form an integral part of the present invention and anycharacteristic that appears novel relative to any prior art on the basisof the description taken in its entirety, including the Examples, formsan integral part of the invention in its function and in its generalapplicability.

Each Example thus has a general scope.

Furthermore, in the Examples, unless stated otherwise, all thepercentages are given by weight, the temperature is expressed in degreesCelsius and the pressure is atmospheric pressure.

EXAMPLES Example 1 Cloning of NRAGE by the Yeast Two-Hybrid Technique

The invention initially consisted in searching for potential partnersfor LOX in keratinocytes. The yeast two-hybrid technique (Y2H) wasemployed. The Y2H system makes it possible to identify and characterizeinteractions between a “lure” protein and “target” potential partners.In the present case, the lure was the mature region of LOX fused to theDNA binding domain (BD) of the Ga14 gene. The target or targets were thegenes encoded by a complementary DNA library of human keratinocytes,fused to the activation domain (AD) of the Gal4 gene.

Interaction of the domains of the lure (LOX) with the domains encoded bya library sequence allows binding and activation of the Gal4 promoter,which controls different genes that confer auxotrophy to AH109 yeasts,enabling growth on deficient medium and activation of the galactosidaseactivities. A gene coding for a protein that was a candidate partner forLOX was thus identified by virtue of this technique for the possibleselection of interactants: the intracellular protein Melanoma AssociatedAntigen D1 (MAGE-D1) or NRAGE (FIG. 1).

Screening of a Library of Keratinocytes by the Yeast Two-HybridTechnique Using the Lure GAL4 BD-hLOXmat (Human LOX Mature)

The cDNA library of normal human skin keratinocytes in vector pGAD-10was used. The lure chosen for this screening was LOXmat encoded by thehuman cDNA region of the LOX enzyme without the signal peptide or thepro-region. The nucleotide sequence was inserted in phase behind the DNAbinding domain (BD) of the GAL4 transcription factor in vector pBD-Gal4Cam. The LOX fragment +494 to +1254 to be inserted was amplified by PCR(polymerase chain reaction), and inserted behind the Gal4 binding domainin vector pGal4-BD, using the following primers:

1) (introducing Bam HI) ggg atc cgc atg gtg ggc gac gac2) (introducing Sal I at the stop which is conserved)ttg tcg act aat acg gtg aaa ttg tgc.

The amplified fragment was initially introduced into vector TOPO andthen inserted into pGa14-BD. To validate the expression of the BD-LOXmatfusion protein, the AH109 yeasts were transformed by lure plasmidpBD-LOXmat.

The transformed yeasts are screened: 6.88×10⁶ cfu (growth in deficientmedium) in the AH109 yeast, 80 selected clones growing in adenine,histidine, tryptophan and leucine deficient medium, 23 of which wereretained for their strong growth.

Example 2 Validation of the Interaction Between LOX and NRAGE by theMammalian Two-Hybrid Technique (M2H)

Hela cells are transfected on the one hand by plasmids pAct andpAct-NRAGE and on the other hand by pBind and pBind-LOXmat in thepresence of lipofectamine. The luciferase activity is tested after 48 hand the results are expressed as the ratio of luciferase activity tocontrol (empty pBind vector). The experiments are performed intriplicate; the mean of the results obtained is shown in FIG. 2.

The hLOX sequence used in Example 1 for yeast two-hybrid screening wasinserted in phase behind the Gal4 binding domain in the pBind vector(Promega, Madison, USA) for the mammalian two-hybrid interactions.

The NRAGE prey was inserted behind the VP16 Gal4 activation domain inthe pAct vector (Promega). It starts at amino acid 152 (nucleotide 458).

Example 3 Co-Immunoprecipitation of LOX and NRAGE in Mammalian Cells

Cos7 cells (mammalian kidney epithelial cells) are co-transfected forthe LOX genes (human complete, human mature region and murine matureregion) by the construct pcLOX32-V5His (LOX), pcLOX36-V5His orpcLOX27-V5His and for the NRAGE gene by pNM3-HA (complete NRAGE) orpNM7-HA (IRD region). The transfection is performed in Petri dishes 100mm in diameter using lipofectamine. After 48 h the transfected cells arelyzed in 500 μl of lysis buffer. The proteins of the cell lyzates areincubated with either anti-V5 or anti-HA; the anti-V5 or anti-HAmonoclonal antibody is added to the cell lyzate at a rate of 1/250 over1 h 30 min at 4° C., with shaking.

The immune complexes (ICs) formed in this way are precipitated withprotein G-Sepharose over 1 h at 4° C., with shaking. After three10-minute washes in lysis buffer and elution in SDS-PAGE buffer, all theICs are subjected to electrophoresis on a 10% SDS-PAGE gel, followed byWestern blotting, Transfer of the ICs to a PVDF (polyvinylidenefluoride) membrane is followed by development:

for NRAGE, with anti-HA diluted to 1/1000, then anti-mouse-HRP (horseradish peroxidase) diluted to 1/20,000,

for LOX, with anti-VS-HRP diluted to 1/5000.

The final detection is effected by chemoluminescence of the HRP.

The results obtained demonstrate that the complete form of NRAGE(NM3-HA) co-immunoprecipitates with the human complete form of LOX (LOX32H), mature human LOX (LOX 36H) and mature murine LOX (LOX 27H).Furthermore, NRAGE (NM7-HA), which corresponds to the IRD region,co-immunoprecipitates in the same way with these three recombinantproteins, showing the involvement of this region in the interaction.

Example 4 Identification, by Immunohistochemistry, of the Presence ofLOX and NRAGE in a Model of Reconstructed Skin

The identification was effected in a model of reconstructed skin(MIMESKIN®, Engelhard Lyon, France) prepared from a dermal substrate(collagen/glycosaminoglycans/chitosan, MIMEDISC®, Engelhard Lyon,France) inoculated with normal human fibroblasts, on whose surfacenormal human keratinocytes were deposited.

After 45 days of culture, allowing differentiation of the keratinocytesby exposure at the air-liquid interface, the samples are fixed inBouin's fixative or formaldehyde and then included in paraffin.

Sections are immunolabeled with the antibodies described below:

anti-LOX antibody obtained and purified by the method described bySommer et al. (Transient expression of lysyl oxidase by livermyofibroblasts in murine schistosomiasis, Laboratory Investigation, 69,460-470, 1993),

anti-NRAGE antibody (goat polyclonal IgG),

rabbit anti-goat secondary antibody.

The immune complexes are detected with a peroxidase-conjugatedanti-rabbit IgG using diaminobenzidine as substrate, followed bycounterstaining with hematoxylin.

FIG. 3 shows the immunohistochemical detection of LOX and NRAGE on thereconstructed skin.

The position of the dermo-epidermal junction is indicated by acontinuous line, the position of the dermal substrate is indicated by anarrow and the location of the keratinocytes is indicated by anarrowhead.

The expression of LOX appears from the basal cell layer upwards,persists in the suprabasal cell layers and progressively disappears inthe differentiated layers. The expression of NRAGE is slightly shifted;it asserts itself from the non-proliferative suprabasal cell layersupwards and intensifies greatly in the granular and horny layers, whereLOX is no longer expressed.

These proteins are therefore associated in the non-proliferativesuprabasal cell layers of the epidermis of the MIMESKIKN® model ofreconstructed skin. This co-location was confirmed by immunohistology(IH) on normal human skin.

The inventors thus demonstrated that the LOX and NRAGE proteins areexpressed in the epidermis, with a co-location zone in the prickle celllayers.

Example 5 Identification of the Co-Location of LOX and NRAGE in NormalHuman Skin by Confocal Microscopy

Frozen sections of samples of normal human skin originating fromsurgical resection (foreskin) are prepared. The anti-LOX and anti-NRAGEprimary antibodies of Example 4 were used to detect the expression ofLOX and NRAGE. The secondary antibodies used are:

donkey anti-rabbit IgG-F1TC, green fluorescein labeling,

donkey anti-goat IgG-R, red rhodamine labeling.

A negative control was prepared in the absence of primary antibodies.

The dual labeling was observed using a ZEISS AXIOPLAN 2 LSM510 confocalupright microscope and the images were acquired using ZEISS LSM5 ImageBrowser software.

FIG. 4 shows the immunodetection of LOX and NRAGE in normal human skinby confocal microscopy.

The observations confirm the results of Example 4, further illustratingthe co-location of LOX and NRAGE in the prickle cell layers of theepidermis of no mal human skin, with an almost perfect juxtaposition ofthe expression of LOX and NRAGE. Thus the observations at the cellularlevel show that LOX and NRAGE appear at the periphery of the cell (inthe submembranous peripheral zone), NRAGE also appearing in thecytoplasm.

The inventors thus demonstrated fulfilment of the conditions for thedirect interaction they had identified in vitro to be able to take placein the epidermis in the zone where both LOX and NRAGE are located.

Example 6 Identification of the Location of LOX and NRAGE in the Skin ofPersons of Different Ages

An immunohistological study using the protocol described in Example 4was carried out on sections of human skin originating from 2 donors fromdifferent age groups (under 20 years and over 60 years).

FIG. 5 shows the labeling performed on the skin of a 91-year-old donor.The observations show a hypoproliferative epidermis that is very thin(reduced to a few cell layers) and hyperkeratinized.

The inventors identified the total absence of LOX (detectable by thetechnique used). On the other hand, NRAGE is strongly expressed, islocated in the cytoplasm and appears from the first suprabasal celllayer upwards, without an expression gradient.

Example 7 Detection of LOX and NRAGE in the Skin of a Person SufferingFrom Graft-Versus-Host Reaction (GVH)

An immunohistological study using the protocol described in Example 4was carried out on sections of human skin from patients suffering fromgraft-versus-host reaction (GVH). This study involved 5 differentdonors.

FIG. 6 identifies an almost total disappearance of LOX in the epidermis(without modification of its expression in the dermis) and a verypronounced presence of NRAGE, which is located in the cytoplasm andappears from the first suprabasal cell layers upwards.

Example 8 Detection of LOX and NRAGE in the Skin of a Person Presentingwith a Cancer of Basocellular and Spinocellular Type

An immunohistological study using the protocol described in Example 4was carried out on samples of skin from patients presenting with abasocellular or spinocellular cancer.

FIG. 7 shows the following in both types of cancer studied:

a progressive decrease in the expression of LOX and NRAGE in theepidermis at the periphery of the tumors,

the absence of LOX and NRAGE in the epidermal invasive cells,

a strong expression of LOX in the dermal stromal reaction around thetumors,

the absence of NRAGE in the dermal stromal reaction around the tumors.

Example 9 Detection of LOX and NRAGE in the Skin of a Patient SufferingFrom Lichen Planus

An immunohistological study using the protocol described in Example 4was carried out on sections of skin from 3 patients suffering fromlichen planus.

FIG. 8 shows a very large decrease in the expression of LOX in theepidermis, or even its total absence. This disturbance correlates withan irregularity in the expression of NRAGE in the epidermis.

Example 10 Identification of the Location of LOX and NRAGE in the Skinof a Patient Suffering From Psoriasis

An immunohistological study using the protocol described in Example 4was carried out on sections of skin from 5 patients suffering frompsoriasis.

FIG. 9 is representative of all the sections observed and shows a verystrong expression of LOX and a moderate presence of NRAGE in theepidermis, with a more or less homogeneous labeling of the zones inquestion, showing no expression gradient. These characteristics of anintensity of expression varying from the norm are coupled with much moreimportant anomalies affecting the location of the proteins in question.Thus, in psoriatic skin, LOX is expressed essentially in the lower partof the epidermis and NRAGE solely in its upper part, the expression ofthe proteins thus being shifted without the overlap zone normallyobserved in healthy skin. At the cellular level, NRAGE is observed onlyin the cytoplasm and not in the submembranous peripheral zone.

Example 11 Identification of the Location of LOX and NRAGE in the Skinof a Patient Suffering From Eczema

An immunohistological study using the protocol described in Example 4was carried out on sections of skin from patients suffering from eczema.

FIG. 10 shows a very strong pericellular expression of LOX in theepidermis. On the other hand, NRAGE is weakly expressed and is observedonly in the cytoplasm, which reflects a loss of co-location at thecellular level.

Example 12 Identification of the Involvement of LOX in the Inhibition ofApoptosis

The study was carried out on monolayer cultures of differentiated humankeratinocytes at confluence. The effect of LOX on apoptosis wasidentified by inhibiting its activity through the addition of β-APN(0.02% w/v—(weight/volume)) under normal or proapoptotic conditions(thermal shock caused by exposure to a temperature of 45° C.±0.5° C. for1 h 30 min).

Cell death by apoptosis is detected and quantified using the TUNEL(terminal deoxynucleotidyl transferase mediated dUTP nick end labeling)technique, which is based on labeling of the DNA deletions accompanyingapoptosis.

The first step consists in labeling the DNA breaks with TdT (terminaldeoxynucleotidyl transferase), which catalyzes the polymerization of thefluorescein-labeled nucleotides at the free 3′-OH end of the DNA. Thesecond step consists in detecting the incorporated fluorescein with analkaline phosphatase-conjugated anti-fluorescein antibody afterincubation with the substrate for the latter.

The experiment is validated by means of a positive control obtained byfragmentation of the DNA with a DNase solution, and a negative controlobtained by depositing phosphate buffer.

The results obtained are shown in the Table below.

TABLE 1 Without thermal shock With thermal shock (% of labeled cells) (%of labeled cells) Without β-APN  6% 39% With β-APN 56% 81%

The results obtained demonstrate that:

thermal shock (45° C.±0.5° C. for 1 h 30 min) induces pronouncedcellular apoptosis, inhibition of the enzymatic activity of LOX by β-APNcauses a substantial increase in apoptosis.

These results identify an antiapoptotic effect of the activity of LOX.

Example 13 Analysis of the Expression of LOX and NRAGE Messenger RNAs byDifferentiated Keratinocytes in Culture in a Calcium Medium, With andWithout Contact With Active Principles Whose Activity is to be Tested(Analysis Performed e.g. by Quantitative RT-PCR, Screening of ActivePrinciples)

The active principles were tested on keratinocytes of normal humanforeskins from young subjects (pooled normal human foreskin epidermalkeratinocytes—Clonetics).

The keratinocytes are amplified e.g. in K-SFM (keratinocyte serum-freemedium), supplemented with antibiotics, up to the third passage at 37°C. under 5% CO₂.

The cells are inoculated into 96-well plates, e.g. at a rate of 40,000cells per cm², and cultivated to about 80% confluence. The cells arethen cultivated in hypercalcium medium (1.7 mM CaCl₂ at 37° C. under 5%CO₂) to induce differentiation of the cells.

The starting material used to prepare the active principles, in the caseof plants (preferably roots, stems, barks, flowers, fruits, seeds,germs, gums, exudates, leaves or whole plants) or proteins, may or maynot be sterilized by radiation, for example beta or gamma radiationpreferably at a dose of 5 kGy, and is then reduced to powder ifnecessary, for example by grinding at room temperature. The powder isthen dispersed at a rate of 2 to 5% (weight/weight) of powder,preferably 5%, either in a polar solvent, e.g. water or butylene glycol,and/or a mixture of polar solvents, advantageously a mixture of waterand an alcohol, glycol or polyol (such as ethanol, glycerol, butyleneglycol and other glycols, xylitol, etc.) in variable proportions, andpreferably a 75/25 or 50/50 water/butylene glycol mixture, or in anapolar solvent, e.g. an alkane, or a mixture of apolar solvents, or in amixture of polar and apolar solvents. After stirring, e.g. magneticstirring, for at least 2 hours, the sample is clarified by decantationor centrifugation and then filtered, preferably on a 0.45 μm or 0.22 μmfilter.

The starting material used to prepare the active principles, in the caseof characterized molecules (e.g. molecules obtained by synthesis orhemisynthesis, biological molecules obtained by purification), isdiluted in a solvent, preferably water or dimethyl sulfoxide (in aconcentration preferably of between 10⁻⁶ M and 10⁻² M and particularlypreferably in the order of 10⁻⁴ M, or preferably of between 1%weight/weight and 5% weight/weight, depending on the molecules). Thesolution obtained is then optionally filtered, preferably on a 0.45 μmor 0.22 μm filter.

The active principles obtained by one of the methods described above arethen tested in a final concentration preferably of between 0.01%volume/volume (v/v) and 10% (v/v) and advantageously of between 0.1% and1% (v/v), for example at 1% (v/v).

Incubation in the presence of the cells is advantageously carried outfor 24 hours in hypercalcium K-SFM without growth factors. The cells arefrozen dry at −80° C. after being rinsed in phosphate buffer pH 7.4.

Extraction of the Total RNAs

The total RNAs are extracted with the SV Total RNA Isolation System(Promega, Meylan, France) for the conditions of 96-well plates accordingto the manufacturer's protocol.

The gene expression is modified by real-time RT-PCR, measuring theexpression of each gene relative to actin (housekeeping gene) andexpressed in % of the untreated negative control.

Quantitative real-time RT-PCR (Q-RT-PCR)

10 μl of total RNAs at 5 ng/μl are added to 40 μl of PCR mix (composedof 25 μl of SYBR Green Buffer Mix 2×, 0.5 μl of enzyme mix, a finalconcentration of 0.5 μM of sense primer and a final concentration of 0.5μM of antisense primer, and RNase and DNase free water qsp 40 μl).

The RT-PCR proceeds in different steps comprising retrotranscription at50° C., 30 min, polymerase activation at 95° C., 15 min, and executionof the PCR cycles (95° C., 15 s; 60° C., 30 s; 72° C., 30 s)×50 cycles.

Production of the Fusion Curve

90° C., 1 min

30° C., 1 min

50° C. to 95° C., 10 s/° C. (fusion curve)

The percentage stimulation or inhibition is expressed relative to theuntreated control (in the absence of test substance).

Actin gene-Hybridization 60° C. Sense GTGGGGCGCCCCAGGCACCA AntisenseCTCCTTAATGTCACGCACGATTTC LOX gene-Hybridization 60° C. SenseACGTACGTGCAGAAGATGTCC Antisense GGCTGGGTAAGAAATCTGATGNRAGE gene-Hybridization 60° C. Sense TGCACAGACATCAGCAGATGG AntisenseTTCACGGATGATATCTCTCAGC Involucrin gene-Hybridization 60° C. SenseTGTTCCTCCTCCAGTCAATACCC Antisense ATTCCTCATGCTGTTCCCAGTGC

To take account of the cell population present, all the results werecompared with the “actin” signal used as a housekeeping gene. Dependingon the experiment, the measurement threshold of the C(T) (=cyclethreshold) was fixed for a value of T of between 0.05 and 0.01, anarbitrary unit of measurement then being calculated for each geneaccording to the following formula:

Sgene <<x>> 10⁷×(1/2)^(C(T)gene <<x>>)

C(T)gene <<x>> denotes the number of cycles necessary to reach thefluorescence threshold of 0.01-0.05 of the gene <<x>>.

The values of the genes of interest were compared with the “actin”signal by calculation of the following ratio:

R=Sgene<<x>>/Sactin

These ratios were compared between the treated and untreated samples,<<x>> being the actin, LOX or NRAGE gene.

Screening of Active Principles

The amounts of cDNA in each assay are compared with the amount of actincDNA and then with the negative controls (without active principles).The results are considered significant if the measured effect reaches afactor of about 2. Of 120 active principles tested, 3 correspond tothese criteria at the concentrations tested and under the conditionsdefined. These active principles form the subject of the Table below:

TABLE 2 NRAGE LOX Name Control multiplied by: Control multiplied by:Ephedra 2 2 Soya 2.5 1 Hops 1 2

Preferably, the ephedra extract is obtained by extraction of the wholeplant, especially with a polar solvent such as water or a water/butyleneglycol mixture (e.g. 75/25 or 50/50), preferably water.

Preferably, the hop extract is obtained by extraction of the cones,especially with a polar solvent such as water or a water/butylene glycolmixture (75/25 or 50/50), preferably water.

Preferably, the soya extract is obtained by extraction of the seed,especially with a polar solvent such as water or a water/butylene glycolmixture (75/25 or 50/50), preferably water.

Conclusions

Of the bank of 120 active principles under the conditions considered:

1 active principle is capable of significantly activating the rate ofsynthesis of mRNA of the genes coding for NRAGE and LOX,

1 active principle is capable of significantly activating the rate ofsynthesis of mRNA of the gene coding for NRAGE, without having an effecton the gene coding for LOX,

and 1 active principle is capable of significantly activating the rateof synthesis of mRNA of the gene coding for LOX, without having aneffect on the gene coding for NRAGE.

This study made it possible to select active principles capable ofmodulating the balance between proliferation, differentiation andapoptosis, at least in the keratinocytes.

The ephedra extract can be used e.g. in the treatment of diseases suchas cancers, preferably cutaneous epithelial cancers (basocellular orspinocellular), or lichen planus, or optionally in the treatment ofcertain cutaneous manifestations of GVH, or else to reduce the effectsof ageing on the skin.

The soya extract can be used e.g. in the treatment of diseases such ascancers, like cutaneous epithelial cancers (basocellular orspinocellular), GVH or lichen planus, or for combating or preventingageing. The soya extract can be used especially for combating cellhyperproliferation.

The hop extract can be used e.g. in the treatment of diseases such ascancers, like cutaneous epithelial cancers (basocellular orspinocellular), eczema or psoriasis. The hop extract can be usedespecially for combating cell hypo-proliferation.

The soya extract and hop extract can be used in association e.g. in thetreatment of diseases such as cancers, like cutaneous epithelial cancers(basocellular or spinocellular), or lichen planus.

The soya extract and ephedra extract can be used in association e.g. inthe treatment of diseases such as cancers, like cutaneous epithelialcancers (basocellular or spinocellular), or lichen planus.

The hop extract and ephedra extract can be used in association e.g. inthe treatment of diseases such as cancers, like cutaneous epithelialcancers (basocellular or spinocellular), or lichen planus.

Example 14 Analysis, by Quantitative RT-PCR, of the Kinetics ofExpression of the Messenger RNAs of NRAGE and LOX on KeratinocytesDuring Calcium Differentiation, and Effect of Active Principles on theExpression Kinetics

The experimental conditions used to obtain cells at 80% confluence areidentical to those described in Example 13. Differentiation takes placein the presence of calcium (1.7 mM CaCl₂) and active principle. Ananalysis is performed by Q-RT-PCR (method described in Example 13) after2, 3 and 4 days of incubation.

Nine other substances were tested in this way. One of them, a cinnamonextract, affords an inhibition of LOX and NRAGE under the experimentalconditions considered.

TABLE 3 NRAGE LOX Name Control multiplied by Control multiplied byCinnamon 2 D 0.9 0.5 3 D 0.7 0.7 4 D 0.2 0.5 (D = Days)

Preferably, the cinnamon extract is obtained by extraction of the bark,especially with a polar solvent such as water or a water/butylene glycolmixture (75/25 or 50/50), preferably water.

Conclusion

The cinnamon extract can be used e.g. in the treatment of diseases suchas psoriasis.

The cinnamon extract and soya extract (whose effect was detected in theprevious Example) can be used in association e.g. in the treatment ofdiseases such as certain cutaneous manifestations of GVH, eczema orpsoriasis, or else for reducing the effects of ageing on the skin.

Example 15 Analysis of the Expression of LOX Messenger RNAs byKeratinocytes in Culture in the Absence of Calcium Differentiation, Withand Without Their Being Put Into Contact With the Active Ingredients,the Activity of Which is to be Tested (an Analysis For Example, Achievedby Quantitative RT-PCR, Screening of Active Ingredients)

The actives were tested on normal human keratinocytes from youngsubjects, obtained by enzymatic extraction of human biopsies collectedafter surgical resection and cultivated as monolayers in a K-SFM(keratinocyte serum free medium with supplements) defined medium,supplemented with antibiotics at 37° C. under 5% CO₂.

The cells are sown at the second passage in 24-well plates, for examplewith an amount of 30 000 cells per cm² and grown until about 95%confluence. The cell carpets are rinsed with phosphate buffer at pH 7.4preferably with calcium and magnesium, before being put into contactwith the active ingredients to be tested or the reference positivecontrols diluted in the K-SFM medium, prepared without supplements butwith antibiotics.

Active ingredients of different origins (vegetable, biotechnologicalorigin or synthesized molecules for example) are tested from 0.1%volume/volume (v/v) to 1% (v/v). An active ingredient from vegetableorigin is tested for example at 1% (v/v) and a synthesized molecule istested for example at 0.1% (v/v)

In particular, the active ingredient from vegetable origin are extractswhich are obtained by macerating plants (preferably roots, rhizomes,stems, barks, flowers, fruit, seeds, germs or leaves) at 2-5% (w/w) in asolvent or a mixture of solvents, advantageously a 100:0 to 0:100water/(alcohol, glycol or polyol) (such as ethanol, glycerol, butyleneglycol and other glycols, xylitol, etc . . . ) mixture. The obtainedextracts are then filtered or distilled in order to recover the solublefraction which is then filtered at 0.45 μm preferably. Thebiotechnological hydrolysates are obtained by fermentation of vegetableextracts in the presence of microorganisms, advantageously from theLactobacillus or Saccharomyces family. These hydrolysates are thenpreferably filtered to 0.45 μm.

The incubation is advantageously performed for 24 hours at 37° C. under5% CO₂, in a K-SFM medium without growth factors but with antibiotics.The negative controls are either the culture medium by itself, or theculture medium containing from 0.1% (v/v) to 1% (v/v) of the solventused during the method for extracting the tested extracts. The referencepositive control used for inducing cell differentiation is a solution ofcalcium chloride (CaCl₂ - final concentration of 1.7 mM).

The untreated cells (NT control) are dry frozen at −80° C. after rinsingwith pH 7.4 phosphate buffer. After treatment for 24 hrs in the presenceof actives or controls, the cells are dry frozen at −80° C. afterrinsing with pH 7.4 phosphate buffer.

Extraction of Total RNAs

The total RNAs are extracted by means of the SV total RNA isolationsystem (Promega, Meylan, France) for the conditions of 24-well platesaccording to the protocol of the manufacturer.

Modification of the expression of the genes is achieved by real timeRT-PCR which measures the expression of each gene relative to actine(housekeeping gene) and expressed as a % of the untreated negativecontrol (NT).

Quantitative Real Time RT-PCR (Q-RT-PCR)

10 μl of 5 ng/μl total RNAs are added to 40 μl of PCR mix (consisting of25 μl of SYBR Green Buffer Mix 2×, 0.5 μl of enzyme mix, final 0.5 μM ofsense primer and final 0.5 μM of antisense primer, RNAse-free andDNase-free water qsp 40 μl).

RT-PCR is performed in different steps including retrotranscription at50° C., 30 min, activation of polymerase at 95° C., 15 min, performingPCR cycles, (95° C.-15 s, 60° C.-30s, 72° C.-63s, 78° C.-30s)×50 cycles.

Producing the Melting Curve

90° C., 1 min

30° C., 1 min

50° C. to 95° C., 10 s/° C. (melting curve)

Primers Used:

Actine gene-hybridization at 60° C. Sense GTG GGG CGC CCC AGG CAC CAAntisense CTC CTT AAT GTC ACG CAC GAT TTC LOX gene-hybridization at 60°C. Sense ACG TAC GTG CAG AAG ATG TCC AntisenseGGC TGG GTA AGA AAT CTG ATG

In order to take into account the present cell population, all theresults are relative to the “actine” signal, used as a housekeepinggene. According to the experimentation, the measuring threshold of C(T)(=cycle threshold) was set for T between 0.05 and 0.01 and then anarbitrary measuring unit is calculated for each gene according to theformula:

Sgene “LOX” 10⁷×(1/2) C(T)gene “LOX”

C(T)gene “LOX” meaning the number of cycles required for reaching thefluorescence threshold of 0.01-0.05 of the “LOX” gene.

The values of the genes of interest were relative to the “actine” signalby calculating the ratio:

R=Sgene“LOX”/Sactine.

These ratios were compared between the treated and untreated samples.

Screening of Active Ingredients:

The cDNA amounts of each test are relative to the amount of cDNA ofactine and then to the negative controls (NT). The results areconsidered as significative when the measured effect is a modulation bya factor of about 2 (stimulation) or 0.5 (inhibition). On 60 testedactives, 30 correspond to these criteria under the defined conditions.These actives are the following and are the subject of the Table hereinbelow:

TABLE 1 LOX modulation vs Preferred part Description Latin name NT ofthe plant Gypsophila Gypsophila 0.4 Root Red sandalwood Pterocarpussantalinus 0.4 Total wood Common bryony Bryonia dioica 0.5 RootButcher's broom Ruscus aculeatus 2.0 Root Lemon Citrus limonia 2.0 FruitTangerine Citrus reticulate 2.1 Fruit Ethyl trans 3-hexanoate 2.3 —Khella Amni Visnaga 2.4 Fruit Alginic acid 2.4 — Carrot Daucus Carota2.4 Root Methyl 2 methyl butyrate / 2.5 — Star anise Illicium verum 2.5Fruit Cypress Cupressus sempervirens 2.6 Fruit Asae Foetida gum 2.6 —Xylitol / 2.8 — Hops Humulus Lupulus 2.8 Fruit Blackthorn Prunus spinosa3.1 Fruit Strawberry tree Arbutus unedo 3.3 Leaf Umbellate wintergreenChimaphila umbellata 3.4 Plant Sweet woodruff Asperula odorata 4.7 PlantMugwort Artemisia vulgaris 4.7 Root Common elderberry Sambucus nigra 5.0Fruit Chinese cabbage Brassica Brassica campestris 5.3 Plant var.Pekinensis Cinnamon Cinnamomum spp 5.5 Stem, bark Chinese ephedraEphedra sinica 6.0 Plant Bitter ash Cassia amara 6.2 Total wood Cacaotree Theobroma cacao 7.4 Fruit shell Silk Serica 7.9 — Red sarsaparillaSmilax ornate 9.0 Root Redcurrant Ribes rubrum 9.1 Fruit PellitoryAnnacyclus pyrethrum 9.8 Root Sweet fennel Foeniculum 10.2 Plant

Conclusions

From the bank of 60 actives and under the considered conditions:

-   -   3 actives are capable of significatively inhibiting the        synthesis rate of mRNA of the LOX coding gene,    -   28 actives are capable of significatively activating the        synthesis rate of mRNA of the LOX coding gene,

With this study, it was possible to select active ingredients capable ofmodulating at least at keratinocyte level, the equilibrium betweenproliferation, differentiation and apoptosis.

Example 15 Analysis of the Expression of Proteins Involved in theRegulation of Epidermal Differentiation/Proliferation Homeostasis in aReconstructed Skin Model With and Without Contact With ActiveIngredients, the Activity of Which is to be Tested by Histology (Exampleof Ephedra Extract).

Demonstration of proliferation or differentiation labels was achieved ina reconstructed skin model (MIMESKIN®, Engelhard Lyons, France) preparedfrom a dermal substrate (collagen/glycosaminoglycans/chitosan,(MIMEDISK®, Engelhard Lyons, France) sown with normal human fibroblasts,at the surface of which normal human keratinocytes were deposited, thecells being extracted by enzyme treatment from biopsies obtained bysurgical resection.

The reconstructed skin model is in particular achieved according to thefollowing protocol:

-   -   0.5 to 1.10⁶ fibroblasts of normal human skin are sown on a        matrix substrate based on collagen/glycosaminoglycan/chitosan,        and then grown in a nutritive medium, for example DMEM-Glutamax        supplemented with 10% of calf serum; ascorbic acid, preferably        at a final concentration of 1 mM; EGF (epidermal grow factor),        preferably at a final concentration of 10 ng/ml; Normocin,        preferably at a final concentration of 100 μg/ml, for 21 days.    -   0.5 to 1.10⁶ fibroblasts of normal human skin are sown on the        dermal equivalent, and then grown in a nutritive medium, for        example DMEM-Glutamax/Ham F-12 (ratio 3:1 v/v) supplemented with        calf serum, ascorbic acid, preferably at a final concentration        of 1 mM; EGF (epidermal growth factor), preferably at a final        concentration of 10 ng/ml; hydrocortisone, preferably at a final        concentration of 0.4 μg/ml; umuline, preferably at a final        concentration of 0.12 IU/ml; isuprel, preferably at a final        concentration of 0.4 μg/mg; triiodothyronine, preferably at a        final concentration of 2.10⁻⁹M; adenine, preferably at a final        concentration of 24.3 μg/ml; Normocin, preferably at a final        concentration of 100 μg/ml. The culture was continued for 7 days        in an immersed condition. The cultures were then placed at the        air-liquid interface for 14 additional days in the same medium        as the immersion culture, except for the calf serum,        hydrocortisone, isuprel, triidothyronine and umuline.

The active ingredient (ephedra extract) is advantageously diluted in theculture media described above at 0.5 and 1% and used 3 days after therespective sowing of fibroblasts and keratinocytes (i.e. days 3 to 21and days 24 to 42). The positive control is preferably made by addingcalcium chloride with a final concentration of 1.5 mM during the emergedphase of the reconstructed skins (i.e. day 28-42) in order to stimulateepidermal differentiation.

At the end of the culture, the samples are frozen, included into athermosensitive resin, and then cryo-cut at 5 μm.

The labellings on the cuts were carried tout with the reagents describedbelow:

-   -   human primary anti-transglutaminase antibody    -   human primary anti-cytokeratin 10 antibody    -   alexa-fluor coupled secondary antibodies    -   Evans blue    -   Dapi

Viewing of immunolabellings is performed under photon microscopy(Axioskop2plus—Zeiss, Germany) and quantification of thetransglutaminase immunolabellings was performed by image analysis(Lucia—Nilon, France) and the effects of the treatment with the activeingredient are evaluated (Holm-Sidak statistical test, p<0.01).

TABLE 1 Quantification of the labelling intensity of transglutaminaseINTENSITY Untreated Calcium Ephedra 0.5% Ephedra 1% 0.149 0.061 0.0820.276 0.099 0.076 0.126 0.196 0.092 0.064 0.094 0.192 Average 0.1130.067 0.100 0.221 Standard deviation 0.031 0.008 0.023 0.048 Variationvs NT 100%  59%  88% 196% Significance vs NT — No No Yes Variation vs Ca169% 100% 149% 330% Significance vs Ca No — No Yes

These results show that the active ingredient induces the same type ofepidermal differentiation as the positive control with a dose-effectrelationship. Indeed, the treated skins have a larger number ofkeratinocyte layers expressing transglutaminase, in particular in theso-called granular layer. Moreover, the obtained labelling is moreintense and more defined as illustrated in the magnification of theepidermal portion. Quantification clearly demonstrates induction of theprotein form after treatment, in particular at 1% active ingredientconcentration (2-fold induction).

FIG. 11 shows the global labelling of the reconstructed skin with Evansblue (red epidermal layers, blue cells, red fibers of the dermalsubstrate, dermo-epidermal junction in dotted lines).

These results show that the active ingredient induces the same type ofepidermal differentiation as the positive control with a dose-effectrelationship. Indeed, the treated skins have a larger number ofdifferentiated keratinocyte layers and in particular, an increase in thethickness of the so-called granular layer is noted. Moreover, aninteresting effect was also obtained on the fibroblast density at thedermas treated with ephedra extract.

FIG. 12 shows the immuno-histochemical detection of cyto-keratin 10 onthe reconstructed skin (cells strongly expressing the red label, thenuclei in blue, the dermo-epidermal junction in dotted lines).

These results show that the active ingredient induces the same type ofepidermal differentiation as the positive control with a dose-effectrelationship. Indeed, the treated skins have a larger number ofkeratinocyte layers expressing cytokeratin 10 and in particular in theso-called granular layer.

FIG. 13 shows the immuno-histochemical detection of transglutaminase onthe reconstructed skin (cells strongly expressing the red label, thenuclei in blue, the dermo-epidermal junction in dotted lines).

Example 17 Use of the Products of the Invention in Cosmetic orPharmaceutical Formulations of the Oil-In-Water Emulsion TypeFormulation 17a:

A Water qsp 100 Butylene Glycol 2 Glycerol 3 Sodium Dihydroxycetyl 2Phosphate, Isopropyl Hydroxycetyl Ether B Glycol Stearate SE 14Triisononanoin 5 Octyl Cocoate 6 C Butylene Glycol, Methylparaben, 2Ethylparaben, Propylparaben, pH adjusted to 5.5 D Products of theinvention 0.01-10%

Formulation 17b:

A Water qsp 100 Butylene Glycol 2 Glycerol 3 Polyacrylamide,Isoparaffin, 2.8 Laureth-7 B Butylene Glycol, Methylparaben, 2Ethylparaben, Propylparaben Phenoxyethanol, Methylparaben, 2Propylparaben, Butylparaben, Ethylparaben Butylene Glycol 0.5 D Productsof the invention 0.01-10%

Formulation 17c:

A Carbomer 0.50 Propylene Glycol 3 Glycerol 5 Water qsp 100 B OctylCocoate 5 Bisabolol 0.30 Dimethicone 0.30 C Sodium Hydroxide 1.60 DPhenoxyethanol, Methylparaben, 0.50 Propylparaben, Butylparaben,Ethylparaben E Perfume 0.30 F Products of the invention 0.01-10%

Example 18 of the Invention Use of the Products of the Invention in aFormulation of the Water-In-Oil Type

A PEG-30 dipolyhydroxystearate 3 Capric Triglycerides 3 CetearylOctanoate 4 Dibutyl Adipate 3 Grapeseed Oil 1.5 Jojoba Oil 1.5Phenoxyethanol, Methylparaben, 0.5 Propylparaben, Butylparaben,Ethylparaben B Glycerol 3 Butylene Glycol 3 Magnesium Sulfate 0.5 EDTA0.05 Water qsp 100 C Cyclomethicone 1 Dimethicone 1 D Perfume 0.3 EProducts of the invention 0.01-10%

Example 19 of the Invention Use of the Products of the Invention in aFormulation of the Shampoo or Shower Gel Type

A Xanthan Gum 0.8 Water qsp 100 B Butylene Glycol, Methylparaben, 0.5Ethylparaben, Propylparaben Phenoxyethanol, Methylparaben, 0.5Propylparaben, Butylparaben, Ethylparaben C Citric acid 0.8 D SodiumLaureth Sulfate 40.0 E Product of the invention 0.01-10%

Example 20 of the Invention Use of the Products of the Invention in aFormulation of the Lipstick Type and Other Anhydrous Products

A Mineral Wax 17.0 Isostearyl Isostearate 31.5 Propylene GlycolDipelargonate 2.6 Propylene Glycol Isostearate 1.7 PEG-8 Beeswax 3.0Hydrogenated Palm Kernel Oil, 3.4 Glycerides, Hydrogenated PalmGlycerides Lanolin Oil 3.4 Sesame Oil 1.7 Cetyl Lactate 1.7 Mineral Oil,Lanolin Alcohol 3.0 B Castor Oil qsp 100 Titanium Dioxide 3.9 CI 15850:10.616 CI 45410:1 0.256 CI 19140:1 0.048 CI 77491 2.048 C Products of theinvention 0.01-5%

Example 20 of the Invention Use of the Products of the invention in anAqueous Gel Formulation (Eye Contour Gel, Slimming Gel, Etc.)

A Water qsp 100 Carbomer 0.5 Butylene Glycol 15 Phenoxyethanol,Methylparaben, 0.5 Propylparaben, Butylparaben, Ethylparaben B Productsof the invention 0.01-10%

Example 21 of the Invention Use of the Products of the Invention in aFormulation of the Triple Emulsion Type

Primary emulsion W1/O A PEG-30 dipolyhydroxystearate 4 CapricTriglycerides 7.5 Isohexadecane 15 PPG-15 Stearyl Ether 7.5 B Water 65.3C Phenoxyethanol, Methylparaben, 0.7 Propylparaben, Butylparaben,Ethylparaben Secondary emulsion W1/O/W2 A Primary emulsion 60 BPoloxamer 407 2 Phenoxyethanol, Methylparaben, 0.3 Propylparaben,2-bromo-2- nitropropane-1,3-diol Water qsp 100 C Carbomer 15 DTriethanolamine pH 6.0-6.5

Example 22 of the Invention Preparation of Pharmaceutical FormulationsContaining the Product of the Invention Formulation 22a: Preparation ofTablets

A Excipients In g per tablet Lactose 0.359 Sucrose 0.240 B Products ofthe invention* 0.001-0.1 *The product of the invention is obtained e.g.by the extraction process described in Example 13, followed by a dryingstep.

Formulation 22b: Preparation of an Ointment

A Excipients Low density polyethylene 5.5 Liquid paraffin qsp 100 BProducts of the invention* 0.001-0.1 *The product of the invention isobtained e.g. by the extraction process described in Example 13,followed by a drying step.

Formulation 22c: Preparation of an Injectable Formula

A Excipient Isotonic saline solution 5 ml B Products of the invention*0.001-0.1 g *The product of the invention is obtained e.g. by theextraction process described in Example 13, followed by a drying step.

Example 23 Evaluation of the Cosmetic Acceptance of a PreparationContaining the Subject of the Invention

The toxicology tests were performed on the compound obtained accordingto Example 2, incorporated at a concentration of 10% in a 0.5% xanthangel, by carrying out an ocular evaluation in the rabbit, by studying theabsence of abnormal toxicity by means of a single oral administration inthe rat, and by studying the sensitizing power in the guinea-pig.

Evaluation of the Primary Cutaneous Irritation in the Rabbit:

The preparations described above are applied undiluted in a dose of 0.5ml to the skin of 3 rabbits according to the method recommended by theOECD directive relating to the study of “the acute irritant/corrosiveeffect on the skin”.

The products are classified according to the criteria defined by thedecree of Feb. 1, 1982 published in the OJRF of Feb. 21, 82.

The results of these tests afforded the conclusion that the products ofthe invention were classified as non-irritant on the skin.

Evaluation of the Ocular Irritation in the Rabbit:

The preparations described above were instilled pure in a singleadministration of 0.1 ml into the eye of 3 rabbits according to themethod recommended by OECD directive no. 405 of 24 Feb. 1987 relating tothe study of “the acute irritant/corrosive effect on the eyes”.

The results of this test afford the conclusion that the preparations canbe considered as non-irritant on the eyes, in terms of directive 91/326EEC, when used pure or undiluted.

Test on the Absence of Abnormal Toxicity by a Single Oral Administrationin the Rat:

The preparations described were given in a single oral administration ina dose of 5 g/kg body weight to 5 male rats and 5 female rats accordingto a protocol based on OECD directive no. 401 of 24 Feb. 1987 andadapted to cosmetic products.

The LD₀ and LD₅₀ are found to be above 5000 mg/kg. The preparationstested are not therefore classified as dangerous preparations oningestion.

Evaluation of the Cutaneous Sensitizing Potential in the Guinea-Pig:

The preparations described are subjected to the maximization testdescribed by Magnusson and Kligmann, the protocol being in accordancewith OECD directive no. 406.

The preparations are classified as non-sensitizing on contact with theskin.

1. A method for regulating balance between cellular proliferation,differentiation and apoptosis in epithelial cells, comprising applying apharmaceutical or cosmetic composition onto an epidermis in needthereof, wherein said composition comprises an effective amount of: i)at least one active ingredient that modulates expression of the lysyloxidase (LOX) having the amino acid sequence of SEQ ID NO: 1; ii) atleast one active ingredient that modulates expression of the NRAGEhaving the amino acid sequence of SEQ ID NO: 2; or iii) a combination ofthe active ingredient of i) and the active ingredient of ii).
 2. Themethod of claim 1, wherein the epithelial cells are keratinocytes. 3.The method of claim 2, wherein the active ingredient of i) increases theexpression of the lysyl oxidase by about 50% in the keratinocytes. 4.The method of claim 2, wherein the active ingredient of i) decreases theexpression of the lysyl oxidase by about 50% in the keratinocytes. 5.The method of claim 2, wherein the active ingredient of ii) increasesthe expression of the NRAGE by about 50% in the keratinocytes.
 6. Themethod of claim 2, wherein the active ingredient of ii) decreases theexpression of the NRAGE by about 50% in the keratinocytes.
 7. A methodof treating aging skin, comprising topically administering onto the skinan effective amount of at least one substance that modulates expressionof LOX and/or NRAGE in the skin, said substance is selected from thegroup consisting of a Sambucus nigra extract, a Humulus lupulus extract,a Ribes rubrum extract, a Cassia amara extract, and an Arbutus unedoextract.
 8. A method for treating disorders relating to epidermalhypoproliferation, comprising topically applying an effective amount ofa substance that stimulates expression of the LOX having the amino acidsequence of SEQ ID NO: 1 and at least one cosmetically orpharmaceutically acceptable excipient.
 9. The method of claim 8, whereinthe substance is a Sambucus nigra extract, a Humulus lupulus extract, aRibes rubrum extract, a Cassia amara extract, or an Arbutus unedoextract.
 10. The method of claim 8, further comprising topicallyapplying an effective amount of a substance that inhibits expression ofthe NRAGE having the amino acid sequence of SEQ ID NO:
 2. 11. A cosmeticor pharmaceutical composition for modulating the interaction between LOXand NRAGE proteins in an epidermis, said composition comprising at leastone substance selected from the group consisting of a Sambucus nigraextract, a Humulus lupulus extract, a Ribes rubrum extract, a Cassiaamara extract, and an Arbutus unedo extract, and at least onecosmetically or pharmaceutically acceptable excipient.